Bottom Line:
Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset.We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity.Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile.

ABSTRACTCD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69- cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile. Thus, CD4+CD25+69- Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.

fig6: ICOS or CTLA-4 blockade in young BDC2.5 animals leads to rapid diabetes onset and correlates with a disruption of the T regulatory/T effector balance. (A) BDC2.5 mice were treated at 9 and 12 d of age with anti–CTLA-4 or ICOS mAb, indicated by arrow (left). Mice were treated with two doses of anti-ICOS mAb starting at 21, 26, or 35 d of age (arrows); with anti–CTLA-4 mAb starting at day 26; or with controls as described in Materials and Methods (right). Diabetes was monitored as aforementioned. (B) BDC2.5 mice were treated at 21 and 24 d with anti-ICOS mAb or control and killed at 26 d of age before diabetes onset. Pancreatic lesion, PLN, and ILN cells were prepared and stained with anti-CD4, B220, CD25, CD69, and live cell dye. Percentages are shown of pancreatic CD4+CD25loCD69+ cells (CD25loCD69+) or CD4+CD25+CD69− cells (CD25+CD69−), gated on live lymphocytes and B220− cells (n = 5; P < 0.008, CD25loCD69+ cells; P < 0.008, CD25+CD69− cells by pairwise Student's t test). PLN and ILN are not shown because there were no significant differences.

Mentions:
ICOS-specific 7E.17G9.G1 (41) and CTLA-4–specific UC10.4F10.11 (American Type Culture Collection) mAbs were purified from hybridoma culture supernatant by standard methods, either in-house or by Bio-Express or Harlan, and tested for endotoxin levels (<1.4 EU/mg protein). Each lot was titered, and doses were set based on Ab equivalents by flow cytometric activity (unpublished data). 7E.17G9.G1 mAb was injected i.p. in two doses of 50–100 μg at 9 and 12 d, or mice were injected with an equal amount of rat IgG2b isotype control (A95-1; BD Biosciences), rat IgG (Sigma-Aldrich), or PBS; or 400 μg of UC10.4F10.11 anti–CTLA-4 mAb as indicated. For anti-ICOS treatment of older mice, two doses of 200–400 μg 7E.17G9.G1 mAb were injected i.p. in PBS at 21 and 24 d, or starting at 26 or 35 d as indicated. In Fig. 6 A (right), an equal volume of post-in-house mAb elution supernatant was used as a control for endotoxin contamination (contains small amounts of residual 17G9).

fig6: ICOS or CTLA-4 blockade in young BDC2.5 animals leads to rapid diabetes onset and correlates with a disruption of the T regulatory/T effector balance. (A) BDC2.5 mice were treated at 9 and 12 d of age with anti–CTLA-4 or ICOS mAb, indicated by arrow (left). Mice were treated with two doses of anti-ICOS mAb starting at 21, 26, or 35 d of age (arrows); with anti–CTLA-4 mAb starting at day 26; or with controls as described in Materials and Methods (right). Diabetes was monitored as aforementioned. (B) BDC2.5 mice were treated at 21 and 24 d with anti-ICOS mAb or control and killed at 26 d of age before diabetes onset. Pancreatic lesion, PLN, and ILN cells were prepared and stained with anti-CD4, B220, CD25, CD69, and live cell dye. Percentages are shown of pancreatic CD4+CD25loCD69+ cells (CD25loCD69+) or CD4+CD25+CD69− cells (CD25+CD69−), gated on live lymphocytes and B220− cells (n = 5; P < 0.008, CD25loCD69+ cells; P < 0.008, CD25+CD69− cells by pairwise Student's t test). PLN and ILN are not shown because there were no significant differences.

Mentions:
ICOS-specific 7E.17G9.G1 (41) and CTLA-4–specific UC10.4F10.11 (American Type Culture Collection) mAbs were purified from hybridoma culture supernatant by standard methods, either in-house or by Bio-Express or Harlan, and tested for endotoxin levels (<1.4 EU/mg protein). Each lot was titered, and doses were set based on Ab equivalents by flow cytometric activity (unpublished data). 7E.17G9.G1 mAb was injected i.p. in two doses of 50–100 μg at 9 and 12 d, or mice were injected with an equal amount of rat IgG2b isotype control (A95-1; BD Biosciences), rat IgG (Sigma-Aldrich), or PBS; or 400 μg of UC10.4F10.11 anti–CTLA-4 mAb as indicated. For anti-ICOS treatment of older mice, two doses of 200–400 μg 7E.17G9.G1 mAb were injected i.p. in PBS at 21 and 24 d, or starting at 26 or 35 d as indicated. In Fig. 6 A (right), an equal volume of post-in-house mAb elution supernatant was used as a control for endotoxin contamination (contains small amounts of residual 17G9).

Bottom Line:
Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset.We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity.Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile.

ABSTRACTCD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69- cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile. Thus, CD4+CD25+69- Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.